The present invention relates to novel compounds useful as A1 adenosine receptor antagonists.
Adenosine receptors are involved in a vast number of peripheral and central regulatory mechanisms such as, for example, vasodilation, cardiac depression, inhibition of lipolysis, inhibition of insulin release and potentiation glucagon release in the pancreas, and inhibition of neurotransmitter release from nerve endings.
In general, adenosine receptors can be divided into two main classes, A1 receptors which can inhibit, and A2 receptors which can stimulate adenylate cyclase activity. One of the best known classes of adenosine receptor antagonists are the xanthines which include caffeine and theophylline. See e.g., Mxc3xcller et al., J. Med. Chem. 33: 2822-2828 (1990). In general, many of these antagonists often suffer from poor water solubility, and low potency or lack of selectivity for adenosine receptors. Additionally, selective analogues of adenosine receptor antagonists have been developed through the xe2x80x9cfunctionalized congenerxe2x80x9d approach. Analogues of adenosine receptor ligands bearing functionalized chains have been synthesized and attached covalently to various organic moieties such as amines and peptides. Attachment of the polar groups to xanthine congeners has been found to increase water solubility. Nonetheless, such developments have yet to fully address problems associated with potency and selectivity. More recently Jacobson et al. J. Med. Chem. 35: 408-422 (1992) has proposed various derivatives of adenosine and theophylline for use as receptor antagonists. The article discloses that hydrophobic substituents are able to potentially enhance affinity. However, it is also acknowledged that such substituents may result in a decrease in solubility thus rendering the antagonists less soluble in vivo. In confronting these problems, Jacobson et al. indicates that a dipropyl substitution at the 1 and 3 positions of theophylline allows desirable affinity at A1 receptors. It is also stated that substitutions at the 7-position are typically not favorable.
It is an object of the present invention to therefore provide compounds useful as A1 adenosine receptor antagonists which display high potency and affinity levels, along with water solubility.
In one aspect, the present invention provides a compound of the general formula: 
wherein
R1 is selected from the group consisting of C1-C8 alkyl;
R2 is of the formula: 
xe2x80x83wherein n is an integer ranging from 1 to 8; R5 is H or CH3(CH2)p, wherein p is an integer ranging from 1 to 7; and R6 is H, (CH2)mH, or (CH2)mOH, wherein m is an integer ranging from 1 to 8;
R3 is selected from the group consisting of: 
xe2x80x83wherein q is an integer ranging from 1 to 8; D is selected from the group consisting of S, NH, and O and wherein R7 is selected from the group consisting of H, OH, NH2, R9COOH, wherein R9 is an alkylene or alkenylene group having 1 to 8 carbon atoms, and (CH2)tOH, wherein t is an integer ranging from 1 to 8; wherein R11 is selected from the group consisting of xe2x80x94CH2COOH and xe2x80x94CH2xe2x80x94CONH(CH2)wNHZ, wherein w is an integer ranging from 1 to 2 and Z is selected from the group consisting of hydrogen and acetate; and
R4 is of the formula: 
xe2x80x83wherein R8 is selected from the group consisting of H; OH; (CH2)fNH2 wherein f is selected from the group consisting of 0 and an integer ranging from 1 to 8; (CH2)sOH, wherein s is an integer ranging from 1 to 8; and R10COOH, wherein R10 is an alkylene or alkenylene group having 1 to 8 carbon atoms; and r is an integer ranging from 1 to 8.
In a second aspect, the invention provides for assay-type probes of the above compound, wherein the probes are marked or conjugated with radioactive or non-radioactive material.
In a third aspect, the invention provides a pharmaceutically acceptable salt of the above compound.
In a fourth aspect, the invention provides a pharmaceutical composition which comprises the above compound and a pharmaceutically acceptable carrier.
The present invention will now be described more fully hereinafter, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The present invention is directed to a compound of the formula (I): 
R1 is selected from the group consisting of C1-C8 alkyl, preferably C1 to C4 alkyl. For the purposes of the invention, R1 is more preferably C1 or C3 alkyl, and is most preferably C3 alkyl.
R2 is of the formula: 
xe2x80x83wherein n is an integer ranging from 1 to 8, more preferably 1 to 4; R5 is H or CH3(CH2)p, wherein p is an integer ranging from 1 to 7, more preferably 1 to 4; and R6 is H, (CH2)mH, or (CH2)mOH, wherein m is an integer ranging from 1 to 8, more preferably 1 to 4.
R3 is selected from the group consisting of: 
xe2x80x83wherein q is an integer ranging from 1 to 8; D is selected from the group consisting of S, O, and NH; and wherein R7 is selected from the group consisting of H, OH, NH2, R9COOH, wherein R9 is an alkylene or alkenylene group having 1 to 8 carbon atoms, and (CH2)tOH, wherein t is an integer ranging from 1 to 8. The alkylene or alkenylene groups may be substituted or unsubstituted. R9 is preferably CHxe2x95x90CH. R11 is selected from the group consisting of xe2x80x94CH2COOH and xe2x80x94CH2xe2x80x94CONH(CH2)wNHZ, wherein w is an integer ranging from 1 to 2 and Z is selected from the group consisting of hydrogen and acetate.
R4 is of the formula: 
xe2x80x83wherein R8 is selected from the group consisting of H; OH; (CH2)fNH2, wherein f is selected from the group consisting of 0 and an integer ranging from 1 to 8; (CH2)sOH, wherein s is an integer ranging from 1 to 8, more preferably 1 to 4; and R10COOH, wherein R10 is an alkylene or alkenylene group having 1 to 8 carbon atoms; and r is an integer ranging from 1 to 8, more preferably 1 to 4. In the above, R9 and R10 are preferably CHxe2x95x90CH.
The invention may be illustrated below with respect to preferred embodiments. In these embodiments, R3 is of the formula:
xe2x80x94(CH2)qC6H4xe2x80x94R7
In one preferred embodiment, R1 is C3 alkyl; R5 is CH3 (CH2)pwherein p is 1; R6 is (CH2)mOH wherein m is 2; R7 is H; R8 is NH2; f is 0; n is 2; m is 2; q is 1; and r is 2.
In another preferred embodiment, R1 is C3 alkyl; R5 is CH3 (CH2)p wherein p is 1; R6 is H; R7 is NH2; R8 is NH2; f is 0; n is 2; q is 1; and r is 2.
In another preferred embodiment, R1 is C3 alkyl; R5 is CH3 (CH2)p wherein p is 1; R6 is H; R7 is H; R8 is NH2; f is 0; n is 2; q is 1; and r is 2.
In another preferred embodiment, R1 is C3 alkyl; R5 is CH3 (CH2)p wherein p is 1; R6 is H; R7 is H; R8 is selected from the group consisting of (CH2)sOH, wherein s is 2 and R10COOH, wherein R10 is CHxe2x95x90CH; n is 2; q is 1; and r is 2.
In another preferred embodiment, R1 is C3 alkyl; R5 is CH3 (CH2)p wherein p is 1; R6 is H; R7 is selected from the group consisting of R9COOH, wherein R9 is CHxe2x95x90CH and (CH2)tOH, wherein t is 2; R8 is NH2; f is 0; n is 2; q is 1; and r is 2.
The compound of the present invention may form pharmaceutically acceptable salts with both organic and inorganic acid and bases. Examples of suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, ascorbic, maleic, methanesulfonic, and the like. Any of the amine acid addition salts may also be used. The salts are prepared by contacting the free base form of the compound with an appropriate amount of the desired acid in a manner known to one skilled in the art. Examples of suitable bases for salt formation are sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, calcium hydroxide, ammonia, organic amines, and the like. The salts may be prepared by contacting the free acid form of the compound with an appropriate amount of the desired base in a manner known to one skilled in the art.
The invention also provides A1 adenosine receptor antagonist compounds with radioactive or non-radioactive labels. Such labelled compounds are useful as assay-type probes or conjugates, and may be used to obtain quantitative binding measurements of the A1 adenosine receptor antagonist compounds. For the purposes of the invention, xe2x80x9cassay-type probesxe2x80x9d refers to those materials which are useful for enhancing the selectivity of the quantitative analysis of the A1 adenosine receptor compounds of the invention. Examples of such assay-type probes are described in U.S. Pat. No. 5,248,770 to Jacobson et al., the disclosure of which is incorporated herein by reference in its entirety. The probes are highly useful in that they have little adverse effect on the affinity of the compounds of the present invention. Radioactive markers include, but are not limited to, an electric spin marker, a 19F NMR probe, a radioactive 18F isotope marker, a radioactive iodine marker (e.g., 125I), a radioactive 3H marker, tritium, and a complex of a metal atom or a metal ion and a chelating agent. An exemplary metal ion is a radioactive isotope of technetium or indium. An exemplary chelating agent is diethylene pentacetic anhydride.
Various non-radioactive materials may be used in labelling the present A1 adenosine receptor compounds. Numerous examples are presented in U.S. Pat. No. 5,248,770 to Jacobson et al. Biotin is used as a common non-radioactive label for such probes, as described in R. W. Old et al. Principals of Gene Manipulation, 4th ed: 328-331 (1989). To facilitate labelling the compounds with biotin or any other appropriate material, a spacer component may be added to the compound according to an accepted method. Such a method is described in the Jacobson et al. ""770 patent. Exemplary spacer components include, but are not limited to, an oligopeptide, triglycidyl, and N-hydroxysuccinimide ester.
Biotin may be bonded to any suitable linkage provided by substituents on the compound structure in accordance with any accepted and suitable technique. For example, referring to compound (I) as defined herein, biotin may be bonded to the hydroxy group on R6 when the compound contains (CH2)mOH at R6 with m defined herein; to the amino group present on either of R7 or R8 when (CH2)fNH2 is contained at the R8 position, wherein f is defined herein; to the hydroxyl group present as R7 or R8; or to the carboxyl group present when R7 and R8 are R9COOH or R10COOH respectively, with R9 and R10 defined herein. Additionally, the biotin may be bonded to a hydroxyl group present on R8, when R8 is (CH2)sOH with s being defined herein. Biotin may also be bonded to R7, when R7 is (CH2)tOH with t being defined herein. The biotin-labeled probes may be detected through appropriate and known analytical techniques.
Fluorescent dyes may also be employed as a non-radioactive labels and are applied to appropriate locations on the compounds of the invention. Such dyes include, but are not limited to, tetramethylrhodamine, fluorescein isothiocyanate, and mixtures thereof. Other non-radioactive materials include for example, nitrobenzoxadiazole; 2,2,6,6-tetramethyl-piperindinyloxy-4-isothiocyanate; and mixtures thereof.
The invention is also directed to a pharmaceutical composition which includes the compound of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition is particularly useful in applications relating to organ preservation in vivo or in situ, perfusion of an isolated organ either removed or contained within the body (e.g., when an organ is transported for transplantation), cardiopulmonary bypass, perfusion of an extremity or limb, and the like. The compounds may be used in intra-articular, intra-thecal, gastrointestinal, and genital urinary applications, as well as in any cavity or lumen such as, for example, the thoracic cavity or ear canal.
The pharmaceutical composition may be employed, as an example, in oral dosage form as a liquid composition. Such liquid compositions can include suspension compositions or syrup compositions and can be prepared with such carriers as water; a saccharide such as sucrose, sorbitol, fructose, and the like; a glycol such as polyethyleneglycol, polypropyleneglycol, and the like; an oil such as sesame oil, olive oil, soybean oil, and the like; an antiseptic such as p-hydroxy- benzoic acid esters and the like; and a flavor component such as a fruit flavor or a mint flavor. The pharmaceutical composition may also be in the form of powder, pills, capsules, and tablets and can be prepared with various carriers. Suitable carriers include, but are not limited to, lactose, glucose, sucrose, mannitol, and the like; disintegrators such as starch, sodium alginate, and the like; binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, and the like; surfactants such as, for example, fatty acid esters; and plasticizers such as, for example, glycerins. The composition of the present invention is especially useful when applied sublingually. It should be noted that in the preparation of the tablets and capsules, a solid pharmaceutical carrier is used. Advantageously, the pharmaceutical composition may be used in the form of, for example, eye drops or an aerosol.
Other types of pharmaceutical compositions may be employed in the form of a suppository, a nasel spray, and an injectable solution. These compositions are prepared using appropriate aqueous solutions which may include, but are not limited to, distilled water, and saline and buffer additives. Other components may be employed such as organic materials including neutral fatty bases. Additionally, the pharmaceutical composition may be utilized in a transdermal application.
Biopolymers may be used as carriers in the above pharmaceutical compositions. Exemplary biopolymers may include, for example, proteins, sugars, or lipids.
The A1 receptor antagonists of the present invention are particularly useful as, for example, anti-allergenics, CNS stimulants, diuretics, anti-asthmatics, and cardiotonics.
Selective analogues of adenosine receptor antagonists have been developed through the xe2x80x9cfunctionalized congenerxe2x80x9d approach. See e.g., U.S. Pat. No. 4,968,672 to Jacobson et al.; and Jacobson et al., Mol. Pharmacol. 29: 126-133 (1985). In terms of pharmacology, the compounds advantageously display increased affinity at A1 receptor sites relative to former A1 receptor antagonists while simultaneously exhibiting good water solubility.
The foregoing example is illustrative of the present invention, and is not to be construed as limiting thereof.